JP6004549B2 - Method for dissolution test of composition containing digestive enzyme - Google Patents

Description

  The present invention is directed to a method for measuring the amount of digestive enzyme released from a solid composition into an elution solvent by fluorescence spectroscopy. The present invention is further directed to a combined method for measuring both dissolution and enteroresistance of a solid composition comprising pancrelipase.

  Solid pharmaceutical compositions or dosage forms such as tablets or capsules generally consist of a mixture of the active ingredient (s) and excipient (s). The reproducibility for the adsorption of the active ingredient (drug) from the solid composition after oral administration depends on several factors such as the release of the drug from the composition under physiological conditions and the dissolution or solubilization of the drug. Since the release of the drug from the composition and the dissolution or solubilization of the drug are critical properties, the dissolution test is very meaningful in predicting the performance of the drug in vivo. Drug approval agencies such as FDA and EMA often require pharmaceutical companies to determine the drug release characteristics of new pharmaceutical compositions in order to obtain approval. These tests also evaluate the quality between batches of pharmaceutical compositions for the purpose of product approval, exemption from bioequivalence requirements, or to demonstrate requirements for non-recommended bioequivalence requirements. May also be required as a USP quality parameter.

  Various protocols for conducting in vitro dissolution tests have been developed and are routinely applied to both product development and quality control. Drug dissolution testing is primarily performed using the recommended compendia methods and equipment such as the US and European Pharmacopoeias, eg USP34 <711> and EP 7.2, 2.9.3. The elution solvents typically used for such tests are, for example, water and buffers such as phosphate buffer or citrate buffer. Various types of elution devices are commercially available based on various agitation methods and are recognized by the Compendia method. Such devices include paddles, baskets, flow-throughs, and reciprocating cylinders. Although the exact procedures (protocols) and equipment are different, all drug dissolution test methods involve adding the pharmaceutical composition or dosage form to the dissolution medium and adding some agitation to the dissolution medium to facilitate the disintegration and dissolution of the drug under test. Including applying.

  The elution solvent and the detection method for determining the amount of drug released into the elution solvent depend on the chemical nature of the drug (selected), and physical and stability considerations are necessary to make an appropriate selection. Is also very important.

  The tests included in the Pancrelipase Delayed Release Capsule, USP Monograph for the determination of digestive enzymes released from pharmaceuticals in oral dosage forms such as pancrelipase delayed release capsules are based on specific measurements of lipase activity. Such methods require long analysis times and are affected by several drawbacks. The main drawback is the marker-lipase instability in the elution solvent, more precisely in the enteric step buffer (pH 6.0) elution solvent. It is necessary to confirm the degree of lipase degradation and then introduce a correction factor in the elution calculation to account for the loss of lipase activity during the test. Furthermore, the complexity of the method (both reagent / substrate preparation and quantitative analysis) results in very high variability in results and also reduces the reproducibility of results between laboratories / laboratories. In addition, the lipase assay has a narrow linearity range (8-16 USP units / mL), which means that this assay only covers capsule titers in the range of 6,400-12,800 USP UI / capsule, and thus a single This is a major limitation because the unit testing approach cannot be implemented. The long analysis time of this method limits the possibilities of using this method for determining multi-point elution profiles.

  There are no methods / procedures that describe how to overcome these drawbacks in determining the release of digestive enzymes from solid compositions.

  Digestive enzymes such as pancrelipase and other pancreatic enzyme preparations (PEP) may be administered to patients suffering from exocrine pancreatic insufficiency (EPI), and the administration of digestive enzyme supplements makes patients more efficient Can digest food.

  Pancreatic exocrine insufficiency (EPI), estimated by the FDA to affect more than 200,000 Americans, is associated with a physiological disorder that prevents food from being properly digested due to a lack of digestive enzymes made in the pancreas. Such a deficiency of digestive enzymes results in disorders such as nutrient indigestion and malabsorption, leading to malnutrition and consequently other undesirable physiological conditions associated therewith. These disorders are common in people with cystic fibrosis (CF) and other conditions that impair the exocrine function of the pancreas, such as pancreatic cancer, pancreatectomy and pancreatitis. Malnutrition can be life-threatening if left untreated, especially in infants and CF patients, and this disorder can lead to impaired growth, decreased immune response and reduced life expectancy.

  Administration of digestive enzymes such as pancrelipase and other pancreatic enzyme preparations (PEP) can treat at least a portion of EPI. The administered digestive enzyme allows the patient to digest food more efficiently.

  Pancreatic enzymes that have been used to treat EPI to compensate for lost digestive function have been used for over 60 years. Until recently, the use of pancreatic enzymes has not been the subject of current regulatory guidelines governing drug approval based on safety, efficacy and manufacturing controls. Pancreatic enzyme replacement therapy has recently been the subject of US and European regulatory efforts. This effort requires the current drug approval review to continue for commercial trading of commercial pancreatic enzyme preparations. Zenpep (R), Creon (R), and Pancrease (R) are three products that have passed the FDA-defined review and are approved for sale in the United States. Again, various pancreatic enzyme preparations are still available in other regions / countries where similar efforts are underway or not yet implemented.

  Capsules containing digestive enzymes such as pancrelipase have been developed for oral administration. However, if the patient is unable to swallow the capsules, open each capsule and sprinkle the contents on a small amount of food, usually soft acidic food (eg, commercially available applesauce) and orally administer to the patient with a spoon. Also good. Alternatively, such drugs may be administered orally to infants and children using a syringe device containing the contents suspended in a solvent that is easy to administer via the syringe device.

  Pancrelipase products are generally labeled as containing three enzyme classes, lipase, amylase and protease, and their levels or titers are described. These enzymes catalyze the hydrolysis of fat to glycerol and fatty acids, hydrolysis of starch to dextrins and sugars, and hydrolysis of proteins to amino acids and derivatives. Digestion, on the other hand, is a complex process involving many other enzymes and substrates that contribute to the correct digestive function and produce various digest products. Other enzymes included in pancrelipase include, among others, trypsin, carboxypeptidase, elastase, phospholipase and coleesterase, and various cofactors and coenzymes. These substances are made naturally in the pancreas and contribute to the correct digestive function.

  Pancrelipase is typically prepared from porcine pancreatic glands, but other sources, such as US Pat. No. 6,051,220, each of which is incorporated herein in its entirety for all purposes, Those described in US Patent Application Publication No. 2004/0057944, US Patent Application Publication No. 2001/0046493, and International Publication No. 2006044529 may be used.

  Pancreatic enzymes show optimal activity under slightly alkaline conditions close to neutrality. Under gastric conditions, pancreatic enzymes can be inactivated and consequently lose biological activity. Thus, externally administered enzymes generally prevent gastric inactivation and remain unchanged during movement from the stomach to the duodenum. Therefore, it is desirable to coat pancreatic enzymes. Pancreatic lipase is most sensitive to gastric inactivation and is an important enzyme in the treatment of malabsorption. The lipase activity is typically monitored to determine the stability of the enzyme composition containing the lipase. Ortenzi et al. The entire contents of U.S. Pat. No. 7,658,918 issued to U.S. Pat. There, a stable digestive enzyme composition is described, explaining that certain particulate drugs that are administered orally are designed to pass through the patient's stomach and then into the intestine. The administration of appropriate dosages of such particulate drugs to patients, particularly infants and children, should be as accurate as possible.

  Unfortunately, no method has been described that measures the amount of digestive enzyme released from a solid pharmaceutical composition or dosage form with excellent accuracy and sensitivity and is readily available between different laboratories.

  The present invention is directed to a method for measuring the amount of digestive enzyme released from a solid composition into an elution solvent by fluorescence spectroscopy. The present invention is further directed to a combined method for measuring both dissolution and enteroresistance of a solid composition comprising pancrelipase.

Pancrelipase beads (Zenpep® mini-tablets)-Elution profile (mean curve) of protease assay. Pancrelipase beads (Zenpep® mini-tablets)-Dissolution profile (mean curve) of lipase assay. Pancrelipase beads (Zenpep® mini-tablets) by fluorescence spectroscopy-dissolution profile (average curve) of total protein assay. Pancrelipase beads (Zenpep® mini-tablets): Elution profile for enzyme specific measurements and fluorescence determination of total protein content. Pancrelipase beads (Zenpep® mini-tablets): Elution profile for lipase assay and fluorimetric determination of total protein. Pancrelipase beads (Zenpep® mini-tablets): Elution profile for protease assay and fluorimetric determination of total protein content. Elution profile of pancrelipase compositions (Zenpep® and Creon®).

  The present invention is directed to a method for measuring the amount of digestive enzyme released from a solid composition into an elution solvent by fluorescence spectroscopy. The amount is measured as the percent of digestive enzyme released from the solid composition or dosage form or single unit form.

  In another embodiment of the method of the invention, the solid composition is a formulation comprising pancrelipase, and more particularly the enteric pancrelipase composition comprises a pharmaceutically inert excipient. It is.

  In another embodiment, the method comprises (a) releasing a digestive enzyme from a solid pancrelipase composition into an elution solvent; (b) reading fluorescence to measure the amount of digestive enzyme in the solvent; including.

  In another embodiment of the invention, the eluting solvent is water, HCl solution, artificial gastric fluid, buffer solution, artificial intestinal fluid, or an aqueous solution or buffer solution containing at least one surfactant.

  In another embodiment, the elution solvent consists of at least two solvents used over time. In addition, a two-step dissolution test may be performed using this method. The first stage is an acidic stage and the elution solvent is an aqueous solvent having an acidic pH, for example in the range of about pH 1 to about 4.5 or about 1 to about 2, or about pH 1.2. The second step is performed with a second elution solvent that is an aqueous buffer having a pH greater than 5, or a pH of about 5.5 to about 6.8, or a pH of about 6.

  In the method according to the invention, the technique used to detect digestive enzymes released from the composition into the elution solvent is fluorescence spectroscopy.

  Molecules have various states called energy levels. Fluorescence spectroscopy is primarily related to electronic and vibrational states. In general, the species studied have a ground electronic state and a higher energy excited electronic state. There are various vibration states in each of these electronic states. In fluorescence spectroscopy, a chemical species first absorbs a photon and excites from its ground electronic state to one of various vibrational states in the excited electronic state. The molecule then returns again to one of the various vibrational levels of the ground electronic state, releasing photons in this process. If the molecule can return to any of several vibrational levels in the ground state, the emitted photons have different energies and thus different frequencies. The fluorescent reaction of proteins is due to the presence of amino acids containing aromatic moieties (tryptophan, tyrosine or phenylalanine). Protein fluorescence reaction is generally measured using an excitation wavelength of 280 nm. Most of the fluorescence emission is due to excitation of tryptophan residues, with a minor contribution by tyrosine and phenylalanine.

  The fluorometric methods disclosed herein are based on measuring the total protein amount of the enzyme released into the elution solvent from a composition or dosage form containing a digestive enzyme (pancrelipase, API). As used herein, the phrase “total protein” refers to the total protein released by the drug product, ie the total protein present in the starting solid composition such as lipase, protease and amylase. To obtain the released API value directly, it is preferable to use the same lot as the drug product to be tested, while grinding it and pouring it into the same elution solvent to obtain a 100% elution API for use in the present method. Prepare a standard. The dissolution of the batch to be tested is measured as a percentage of the standard.

  The methods of the invention can be applied to any suitable oral dosage form comprising a pancrelipase solid composition that may contain a pharmaceutically inert excipient, such as a digestive enzyme. Non-limiting examples of suitable dosage forms include tablets, capsules, sachets or single unit dosages. In certain embodiments, the dosage form is a capsule. Each dosage form contains API (drug) digestive enzyme beads (also called units). In the present invention, the digestive enzyme beads are any kind of fine particles. The term “bead” includes granules, particles, tablets, spheres, mini-tablets, micro-tablets, micro-particles, microspheres, mini-microspheres, microcapsules and micropellets. The beads may be any suitable particle size or shape, particularly having a size in the range of about 50 to about 5,000 μm, more particularly the beads are in the range of about 2 to about 5 mm, or less than about 2 mm. For example, having a nominal (eg, average) particle diameter of about 1-2 mm. “Mini-microspheres” have a minimum median diameter of 1.15 mm, or “microtablets” have a maximum diameter of 2.63 mm, which are also suitable for this method. The beads may have an average size of less than about 800 μm, preferably less than 500 μm, preferably from about 400 μm to about 600 μm or from about 250 μm to about 500 μm. These beads have a volume particle size of 400 μm or more (d (v, 0.1) (10% of the volume distribution is less than this value and 90% is defined as a diameter greater than this value) and 900 μm or less. Volume particle size d (v, 0.9), (90% of the volume distribution is less than this value and 10% is defined as a diameter greater than this value).

  All digestive enzyme beads suitable for pharmaceutical preparation, more particularly pancrelipase enzyme beads, may be coated with an enteric layer. In embodiments where the pancrelipase base tablet is surrounded by an enteric coating, the coating acts as a barrier, protecting the drug from the acidic environment of the stomach and substantially preventing the release of the drug before it reaches the small intestine. Suitable combinations of enteric coating compositions and other coating compositions may be used to obtain the desired type of control over drug release or therapeutic effect. The enteric coating includes at least one enteric polymer, and further excipients. The phrase “enteric polymer” refers to a polymer that protects digestive enzymes from the stomach contents, such as a polymer that is stable at acidic pH but can be rapidly degraded at higher pH, or other parts of the gastrointestinal tract. In particular, it means a polymer that has a sufficiently slow rate of hydration or corrosion so that contact between the stomach contents and digestive enzymes is relatively small while remaining in the stomach. Non-limiting examples of entero-resistant polymers include cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, copolymers of methacrylic acid, esters of methyl methacrylate and shellac. . These polymers are commercially available under various trade names such as: Cellacefate® (cellulose acetate phthalate), Eudragit® L100, S100, L30D, FS30D, L100-55 (copolymers of methacrylic acid). ), Aquateric® (cellulose acetate phthalate), Aquat® (hydroxypropylmethylcellulose acetate succinate), HP55® (hydroxypropylmethylcellulose phthalate). Preferably the enteric coating is 10-20 wt. % Of at least one enteric polymer. Each wt. % Is based on the total weight of the coated particles. The coating is a lipophilic agent such as a C6-C30 lipophilic low molecular weight molecule selected from aliphatic carboxylic acids and alcohols, preferably C14-C18 carboxylic acids or alcohols such as stearic acid, myristic acid, myristic acid alcohol or stearyl. Alcohol may further be included. Other optional components of the coating include plasticizers, lubricants (eg, talc, magnesium stearate, colloidal silicon dioxide and combinations thereof; and optionally low viscosity ethylcellulose). Non-limiting examples of suitable plasticizers include triacetin, tributyl citrate, triethyl citrate, acetyl tri-n-butyl citrate, diethyl phthalate, dibutyl sebacate, polyethylene glycol, polypropylene glycol, castor oil, acetylated Mono- and di-glycerides, cetyl alcohol, myristyl alcohol and mixtures thereof. A preferred plasticizer is a non-phthalic plasticizer or a mixture thereof.

  The coated stabilized digestive enzyme particles may then be formulated as a capsule. A particular stabilized digestive enzyme particle dosage form is a capsule containing enteric pancrelipase enzyme beads. An enteric coated pancrelipase enzyme comprising hydroxypropyl methylcellulose having a water content of about 6 wt% or less, for example, more specifically having a water content of about 4 wt% or less, and more particularly having a water content of about 2 wt% or less Is a specific embodiment of the formulation.

  As used herein, the term “digestive enzyme” refers to an enzyme in the digestive tract that breaks down the components of food so that the organism can ingest or absorb it. Non-limiting examples of digestive enzymes include pancrelipase (also called pancreatin), lipase, colipase, trypsin, chymotrypsin, chymotrypsin B, pancreatatopeptidase, carboxypeptidase A, carboxypeptidase B, glycerol ester hydrolase, Phospholipase, sterol ester hydrolase, elastase, kininogenase, ribonuclease, deoxyribonuclease, α-amylase, papain, chymopapain, glutenase, bromelain, ficin, β-amylase, cellulase, β-galactosidase, isomaltase and mixtures thereof . These can be obtained by extraction from the pancreas or pancreatic juice, or artificially produced from sources other than the pancreas, such as microorganisms, bacteria, molds, fungi, plants or other animal tissues, genetically modified microorganisms, fungi or plants To do or get.

The term “pancrelipase” or “pancrelipase enzyme” or “pancreatin” refers to a mixture of several types of enzymes, including an amylase enzyme, a lipase enzyme and a protease enzyme, or a mixture of enzymes from the pancreas, etc. . Pancrelipase is commercially available from, for example, Nordmark Arzneimtel GmbH, Scientific Protein Laboratories LLC or Sigma Aldrich, but similar extracts from porcine, bovine or other mammalian sources may be used. Examples of commercially available pancrelipase formulations include Zenpep, Viokace, Ultrase, Creon, Pancrease, and Panzytrat. More specifically, Zenpep capsules for oral administration are enteric beads (1.8-1.9 mm of 10,000, 5,000, 5,000 USP lipase units, 10,000, 15,000, 20,000, 25,000 and 40,000 USP lipase units 2.2-2.5 mm).

The term “lipase” refers to an enzyme that catalyzes the hydrolysis of lipids to glycerol and simple fatty acids. Examples of lipases suitable for the present invention include animal lipases (eg, porcine lipase), bacterial lipases (eg, Pseudomonas lipase and / or Burkholderia lipase), fungal lipases, plant lipases, recombinant lipases. (Eg, those produced by recombinant DNA technology in suitable host cells selected from any one of microbial, bacterial, yeast, fungal, plant, insect or mammalian host cultured cells, or homologous to natural sequences or Recombinant lipases comprising amino acid sequences that are substantially identical, lipases encoded by nucleic acids that are homologous or substantially identical to nucleic acids encoding natural lipases), synthetic lipases, chemically modified lipases, and mixtures thereof Yes, but limited to this Not to. The term “lipid” broadly includes natural molecules such as fats, waxes, sterols, fat-soluble vitamins (eg vitamins A, D, E and K), monoglycerides, diglycerides, triglycerides, phospholipids and the like.

The term “amylase” refers to a glycoside hydrolase enzyme that degrades starch, such as α-amylase, β-amylase, γ-amylase, acid α-glucosidase, salivary amylase, such as ptyalin. Amylases suitable for use in the present invention include animal amylases, bacterial amylases, fungal amylases (eg, Aspergillus amylase, eg Aspergillus oryzae amylase), plant amylases, recombinant amylases (eg, A suitable host cell selected from any one of microorganisms, bacteria, yeast, fungi, plants, insects or mammalian host cultured cells, or produced by recombinant DNA technology, or homologous or substantial to a native sequence Recombinant amylases containing amino acid sequences that are identical in nature, amylases encoded by nucleic acids that are homologous or substantially identical to nucleic acids encoding natural amylases), chemically modified amylases, and mixtures thereof, to this It is not limited.

The term “protease” generally refers to an enzyme (eg, proteinase, peptidase or proteolytic enzyme) that breaks peptide bonds between amino acids of a protein. Proteases are generally distinguished by the type of catalyst, for example, aspartate peptidase, cysteine (thiol) peptidase, metallopeptidase, serine peptidase, threonine peptidase, alkaline or semi-alkaline protease, neutral peptidase whose catalytic mechanism is unknown. Non-limiting examples of proteases suitable for use in the present invention include serine proteases, threonine proteases, cysteine proteases, aspartic proteases (eg, plasmepsin) metalloproteases and glutamate proteases. Furthermore, suitable proteases for use in the present invention include animal proteases, bacterial proteases, fungal proteases (eg Aspergillus meleus protease), plant proteases, recombinant proteases (eg bacteria, yeast, fungi, A suitable host cell selected from any one of plant, insect or mammalian host cultured cells, produced by recombinant DNA technology, or recombinant comprising an amino acid sequence that is homologous or substantially identical to the native sequence Proteases, proteases encoded by nucleic acids that are homologous or substantially identical to nucleic acids encoding natural proteases), chemically modified proteases and mixtures thereof, but are not limited thereto.

  The pancrelipase enzyme in the composition or oral dosage form analyzed in the present invention is one or more lipases (ie, one lipase, or two or more lipases), or one or more amylases. (Ie, one amylase, or two or more amylases), or one or more proteases (ie, one protease, or more than one protease), as well as various combinations of these enzymes and A mixture of ratios may be included.

  The lipase activity in the composition or dosage form analyzed by the methods herein is about 650 to about 45,000 IU (USP method), about 675 to about 825 IU, about 2,700 to about 3,300 IU, about 4 , 500 to about 5,500 IU, about 9,000 to about 11,000 IU, about 13,500 to about 16,500 IU, about 18,000 to about 22,000 IU, about 22,500 to about 27,500 IU, about 36 May range from 4,000 to about 44,000 IU, and ranges and subranges therebetween. The lipase activity is about 750 IU, about 3,000 IU, about 4,200 IU, about 5,000 IU, about 6,000 IU, about 10,000 IU, about 10,500 IU, about 15,000 IU, about 16,800 IU, about 20, The activity may be 000 IU, about 21,000 IU, about 24,000 IU, or about 25,000 IU, or about 40,000 IU (USP method) or multiples thereof. The amylase activity in the composition or dosage form is about 1,600 to about 6,575 IU (USP method), about 6,000 to about 225,000 IU, such as about 6,400 to about 26,300 IU, about 10,700. To about 43,800 IU, about 21,500 to about 87,500 IU, about 32,100 to about 131,300 IU, about 42,900 to about 175,000 IU, about 53,600 to about 218,700 IU, and between these Range and partial range. The protease activity in the composition or dosage form is about 1,250 to about 3,850 IU (USP method), about 5,000 to about 130,000 IU, such as about 5,000 to about 15,400 IU, about 8,400. To about 25,700 IU, about 16,800 to about 51,300 IU, about 25,000 to about 77,000 IU, about 33,500 to about 102,600 IU, about 41,800 IU to about 128,300 IU, and between these Range and partial range. The enzyme combined composition comprises: (A) the lipase activity may range from about 675 to about 825 IU, and the amylase activity may range from about 1,600 to about 6,575 IU. Protease activity may range from about 1,250 to about 3,850 IU (USP method); (B) lipase activity may range from about 2,700 to about 3,300 IU; The activity may be in the range of about 6,400 to about 26,300 IU and the protease activity may be in the range of about 5,000 to about 15,400 IU (USP method); (C) the lipase activity It may range from about 4,500 to about 5,500 IU, amylase activity may range from about 10,700 to about 43,800 IU, and protease activity may range from about 8,400 to about May be in the range of 5,700 IU (USP method); (D) the lipase activity may be in the range of about 9,000 to about 11,000 IU, and the amylase activity is in the range of about 21,500 to about 87, 500 IU, and protease activity may be in the range of about 16,800 to about 51,300 IU (USP method); (E) lipase activity of about 13,500 to about 16,500 IU In which the amylase activity may range from about 32,100 to about 131,300 IU, and the protease activity may range from about 25,000 to about 77,000 IU (USP) (F) the lipase activity may range from about 18,000 to about 22,000 IU and the amylase activity ranges from about 42,900 to about 175,000 IU; Protease activity may range from about 33,500 to about 102,600 IU (USP); and (G) lipase activity may range from about 22,500 to about 27,500 IU. Well, amylase activity may range from about 53,600 to about 218,700 IU and protease activity may range from about 41,800 IU to about 128,300 IU (USP). Also, the lipase activity in the composition or dosage form analyzed by the methods herein may range from about 5,000 PhEur lipase units to about 40,000 PhEur lipase units, and the lipase activity is about 5,000 PhEur. It may be a lipase unit, alternatively about 10,000 PhEur lipase unit, alternatively about 15,000 PhEur lipase unit, alternatively about 20,000 PhEur lipase unit, alternatively about 30,000 PhEur lipase unit, or about 40,000 PhEur lipase unit.

  In another embodiment of the invention, the procedure may further be used to analyze a single unit containing a portion of the amylase activity described above.

  In another embodiment of the invention, the method may further be used to analyze a single unit comprising a portion of the amylase activity described above.

  The ratio of amylase activity / lipase activity in the composition or dosage form may range from about 1 to about 10, for example from about 2.38 to about 8.75 (enzyme assays are performed according to USP). This ratio may range from about 1 to about 8, such as from about 1.86 to about 5.13 (enzyme assay performed according to USP), or the ratio may be about 1 or about 2, It may be about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.

  Inactive ingredients of the product include croscarmellose sodium, hydrogenated castor oil, colloidal silicon dioxide, microcrystalline cellulose, magnesium stearate, hypromellose phthalate, talc and triethyl citrate. Taking a single dose of the preparation of Aptalis Pharma is a very stable formulation that gives patients and physicians a certain amount of the main pancreatic enzyme lipase, protease and amylase. The contents may be separated by opening capsules to individually adjust the dose.

  Another embodiment of the present invention, the method comprises: (a) releasing a digestive enzyme from a solid pancrelipase composition into an elution solvent; (b) reading fluorescence to detect the enzyme and detecting the digestive enzyme in the solvent. Measuring the quantity. Dissolution testing is performed using the dissolution apparatus described in the Compendia USP or EMA method, or using all such apparatus and protocols known and utilized by experts in the field. The elution solvent is selected from a variety of solutions suitable for total protein elution testing, such as water, HCl solution, artificial gastric fluid, buffer solution, artificial intestinal fluid, aqueous solution or buffer containing a surfactant. The buffer may be, for example, a phosphate buffer or a citrate buffer.

  In certain embodiments, the elution solvent consists of at least two elution solvents that are used over time (two stages). The first eluting solvent is an aqueous solvent having an acidic pH of about 1 to 4.5, in particular about 1 to about 2, more particularly about pH 1.2 (acidic stage), An aqueous solution having a pH above about 5.0, in particular a pH of about 5.5 to about 6.8, more particularly a pH of about 6 (buffered enteric phase).

  In another embodiment of the invention, the first elution solvent is an aqueous solvent having a pH of about 1 to about 4.5 and the second elution solvent is an aqueous buffer having a pH of greater than about 5. .

  In another embodiment, the first elution solvent is an aqueous solvent having a pH of about 1 to about 4.5, and the second elution solvent is an aqueous buffer having a pH of about 5.5 to about 6.8. It is.

  In another embodiment of the invention, the first elution solvent is an aqueous solvent having a pH of about 1 to about 2, and the second elution solvent is an aqueous buffer having a pH greater than about 5.

  In another embodiment of the invention, the first eluting solvent is an aqueous solvent having a pH of about 1 to about 2, and the second eluting solvent is an aqueous buffer having a pH of about 5.5 to about 6.8. It is a liquid.

  In another embodiment of the present invention, the first elution solvent is an aqueous solvent having a pH of about 1.2, and the second elution solvent is an aqueous buffer having a pH of about 6.

  In a further embodiment, the method comprises the steps of a) adding a solid pancrelipase composition to the first eluting solvent (acidic phase) and b) transferring the suspension to the second eluting solvent (enteric phase). ), C) causing the release of the digestive enzyme, d) sampling an aliquot of the eluting solvent, e) reading the fluorescence at 346 nm, and d) calculating the amount of released digestive enzyme; including. Calculations are made as reported in the examples.

  When a drug product is subjected to a dissolution test, USP requires calculation of a “Q” value. This Q value correlates with the displayed titer and the current acceptance criteria is fixed at 75% of the displayed lipase activity.

式中、溶出率（％）は放出されたＡＰＩの％であり、分析手順に示したように算出され；バッチリパーゼアッセイはバッチリパーゼ活性であり；表示リパーゼ活性は、薬剤製品のラベルに表示されたリパーゼ活性である。 The fluorescence measurement method of the present invention shows sufficient correlation with the enzyme elution profile even if it is not dedicated to the measurement of enzyme activity, so that the total protein release is closely correlated with the enzyme release. Proved. Thus, the “Q” value of this method can be calculated as follows: Q ”value of elution method by fluorescence measurement:

Where% dissolution is the% of API released and is calculated as indicated in the analytical procedure; batch lipase assay is batch lipase activity; the indicated lipase activity is shown on the drug product label Lipase activity.

  The fluorometric method disclosed herein correlates well with the measurement of enzyme activity while conducting a buffered enteric dissolution test of the pancrelipase composition, thus substituting the lipase activity enzyme test. Although this is considered a method, as in the case of the lipase assay, this method eliminates the gastro-resistance problem that occurs during elution in the acidic phase, since the fluorescence measurement of total protein is not affected by acid membrane permeation. It cannot be detected at all. The lipase activity is strongly reduced when the acidic solution permeates the protective film. In the current USP test (measurement of lipase activity), the action of gastro-resistance, which is considered to be poor in the acidic phase, is dissolved in the buffered enteric phase along with the elution and degradation phenomena of lipase occurring in the buffered enteric phase. Accumulate at the end of.

  Accordingly, another embodiment of the present invention is a method for measuring the amount of digestive enzyme released from a solid pancrelipase composition into an elution solvent by fluorescence spectroscopy in combination with a gastroresistance test. In this method, enteric solubility is measured by determining the residual lipase activity of a product by a specific lipase assay after exposure to an acidic solvent (acid phase). In this embodiment, two tests (elution FL test and enteric GR test) may be performed in turn.

  FL test: Dissolution test performed in two stages (acidic and enteric). The elution solvent for the first acidic stage is an aqueous solvent having an acidic pH of about 1 to about 4.5, preferably about pH 1 to about 2, and preferably about pH 1.2 (acidic stage); In the enteric phase, the elution solvent is an aqueous buffer having a pH above about 5, preferably a pH of about 5.5 to about 6.8, preferably about pH 6 (buffered enteric phase). The entero-resistance test is conducted in an aqueous solvent having an acidic pH of about 1 to about 4.5, preferably about pH 1 to about 2, and preferably about pH 1.2. In the fluorescence test, the digestive enzyme (API or drug) released at the end of the enteric phase is measured. The acceptance criterion Q value can be calculated by the ratio of batch released lipase activity / indicated lipase activity.

  GR test: An enteric test is performed under the conditions of the acidic stage of the dissolution test (the dissolution solvent has an acidic pH of about 1 to about 4.5, in particular a pH of about 1 to about 2, more particularly a pH Measure the entericity (%) by determining the residual lipase activity of the product after exposure to acidic solvent using a lipase assay, which is an aqueous solution of about 1.2, and the acceptance criteria is USP delay Acceptance criteria used for the acid phase of the release dosage form.

  From the foregoing description and experimental section, it can be seen that the present fluorescence analysis method has several important advantages.

  Because the present invention provides a simple and rapid procedure, the time required for reagent preparation is greatly reduced and no specific analytical expertise is required for enzyme assays. For this reason, it is very easy to transfer the analysis method. The proposed method is simpler and faster than performing a lipase assay, and the time required for reagent preparation is greatly reduced.

  The marker (total protein) is stable in the eluting solvent and therefore does not require a correction factor (required for lipase assay) to correct for degradation. In fact, the marker assayed fluorometrically (total protein) is measured using this method, whereas it shows less than 3% degradation after 30 minutes in an enteric phase solvent buffer at 37 ° C., pH 6. The lipase enzyme activity shows about 11% degradation in an enteric phase solvent buffer at 37 ° C., pH 6.

  Also, because this new method is accurate and has excellent sensitivity, the limit of quantification / linearity range is also suitable for single unit testing. The fluorimetric method shows superior performance characteristics over the lipase enzyme assay for a working concentration of 0.3 lipase USP units, about 3 μg pancrelipase / mL, which is about 1/50 of the working concentration of the lipase assay. The linearity range is 10 to 200% of the concentration used, and the accuracy is less than 20% for both the parallel accuracy and the intermediate accuracy (for pancrelipase preparations, six lots of Zenpep®). Measured by elution results).

  Furthermore, using such a method, a multipoint (> 3) test elution profile can also be obtained.

  Also, in the experimental part, the non-specific fluorescence procedure of the present invention for detecting the total protein amount based on the comparison of elution profiles obtained by the three measuring methods is based on two enzyme-specific methods based on protease activity and lipase activity. It is also shown to be equivalent in performance to the assay (current Compendia method).

  It will be understood that both the foregoing summary and the following detailed description are illustrative of the invention and are not intended to limit the invention.

Equipment, Materials and Methods Equipment: LS 50B Fluorescence Spectrometer (Perkin Elmer), LS 55 Fluorescence Spectrometer (Perkin Elmer), Lambda 20 UV-VIS Spectrometer (Perkin Elmer), 786 TitlandoPetrometer 7025 elution tank (Vankel), Premier 5100 elution tank (Distek), USP Apparatus 1-basket (for acidic stage); USP Apparatus 2-paddle (for enteric stage).

  Reagent for dissolution test. Acid stage solvent (pH 1.2): Add 2.00 g of sodium chloride to 800 mL of purified water and stir until completely solubilized. Add 7 mL of 37% HCl and mix. The pH of the solution is adjusted to 1.20 ± 0.05 with 1N HCl or 1N NaOH. Dilute to 1000 mL with purified water, check pH and adjust to 1.20 ± 0.05 with 1N HCl or 1N NaOH if necessary.

  Reagent for dissolution test. Enteric stage solvent (pH 6.0): 9.20 g of monobasic potassium phosphate and 2.00 g of sodium chloride are added to 800 mL of purified water and stirred until completely solubilized. The pH of the solution is adjusted to 6.00 ± 0.05 with 1N NaOH. Dilute to 1000 mL with purified water, check pH and adjust to 6.00 ± 0.05 with 1N HCl or 1N NaOH if necessary.

  All examples are performed using enteric coated pancrelipase beads, pancrelipase mini tablets (MT) or microtablets (MCT). These are pancrelipase raw materials and excipients (eg, croscarmellose sodium, hydrogenated castor oil, colloidal silicon dioxide, microcrystalline cellulose and stearin coated with enteric polymer hypromellose phthalate (HP55). Acid magnesium). These MTs and MCTs are placed in HPMC capsules and are marketed under the name Zenpep®. One skilled in the art will recognize that other enteric polymers and excipients may be used for enteric coated pancrelipase beads.

  Lipolytic activity is measured by a method based on the Compendia procedure of the lipase assay described in the USP monograph of pancrelipase. This procedure is based on titrating free fatty acids formed from hydrolysis of esterified fatty acids using a substrate (olive oil) by the pH stat method. It is based on the principle that lipase catalyzes the hydrolysis of triglycerides and free fatty acids (FFA) are formed. When the FFA formed as a function of time is titrated, the enzyme activity of the lipase is determined, which can be expressed in units, and 1 μmol of FFA is formed per minute in 1U. This reaction is carried out while maintaining a constant pH value by an experimental system in which NaOH (titrant) is added when the pH value changes compared to a fixed value (pH stat method). The amount of titrant added as a function of time corresponds to the amount of FFA formed by the action of lipase on triglycerides. Working under experimental conditions where the enzyme is stable with a suitable amount of substrate, linear kinetics of FFA formation as a function of time can be obtained. The lipase enzyme activity is obtained by the slope of the curve {added titrant = f (amount (mL) / hour (min))}.

  Proteolytic activity is measured according to the Compendia procedure described in the USP monograph of pancrelipase.

Example 1
Preparation of drug product standard solution The standard solution is prepared using the same lot of drug product in the dosage form under analysis. An amount of pancrelipase beads (Zenpep® minitabs or microtabs) equivalent to 7,000 USP lipase units is accurately weighed into a mortar. Add 5-6 mL of enteric stage solvent and grind until complete dispersion of product is obtained. Transfer this suspension to a 500 mL volumetric flask. Rinse the mortar with a few mL of enteric stage solvent 2-3 times and transfer the liquid to a 500 mL volumetric flask. The enteric stage solvent is added to the volumetric flask until the total volume is finally 500 mL and the mixture is stirred for 10 minutes. Aliquots sampled from the eluting solvent are further diluted 1:50 with enteric stage solvent. This dilution results in a final API concentration of about 0.3 USP lipase units (about 3 μg pancrelipase / mL). This final dilution is done with dissolution test-endpoint only and dissolution test-multiple points (dissolution profile).

Example 2
Dissolution test 800 mL of acidic stage solvent is added to each vessel of the dissolution tank equipped with a basket device and the dissolution solvent is equilibrated at 37 ° C. Weight is added to an amount of pancrelipase beads (Zenpep minitabs or microtabs) equivalent to 11,200 USP lipase units (14 USP lipase units / mL), thus preparing six independent samples and placing them in a basket. The apparatus is operated at 100 rpm. After 1 hour, the baskets are removed from the solvent, rinsed with a few milliliters of water, and the contents of each basket are transferred to a corresponding vessel containing 800 mL of enteric-stage solvent at 37 ° C. in an elution tank equipped with a paddle device. . The apparatus is operated at 100 rpm. After 30 minutes, an aliquot of the elution solvent in each vessel is sampled to measure the released API. When determining the elution profile, a 2.5 mL aliquot of the enteric phase elution solvent is sampled at 10, 12, 15, 18, and 30 minutes. The solvent is not changed during the test, and the amount of reduction is taken into account by the following correction factor when calculating.

Example 3
Determination of digestive enzymes (API, active ingredient) released in dissolution test by fluorescence spectroscopy (total protein assay)
Each aliquot of elution solvent sampled from each basket as described in Example 2 is diluted 1:50 with enteric stage solvent. Read the diluted solution with a fluorescence spectrometer with the following operating parameters: quartz cuvette with 1 cm optical path length; excitation wavelength: 280 nm; emission wavelength (measurement): 346 nm, excitation slit: 6.0. Endpoint: The marker target concentration (API = pancrelipase; 100% release) at 0.3 USP lipase units / mL or about 3 μg of pancrelipase / mL is obtained by the following calculation:

  The amount of API released was determined for a standard solution prepared as described in Example 1.

In the dissolution test-endpoint, the calculation is performed using the following formula:

式中、Ｅ_ＳＭＰはサンプルのブランクを差し引いた蛍光測定値（３４６ｎｍの発光）；Ｅ_ＳＴＤは標準のブランクを差し引いた蛍光測定値（３４６ｎｍの発光）；Ｗ_ＳＭＰはサンプル重量（ｍｇ）；Ｗ_ＳＴＤは標準の重量（ｍｇ）；Ｖ_ＳＭＰはサンプの希釈量（ｍＬ）；Ｖ_ＳＴＤは標準の希釈量（ｍＬ）；ＦＣは補正係数である（表１を参照）。

For dissolution test-multi-point (dissolution profile), the calculation is performed using the following formula:

_Where E _SMP is the fluorescence measurement minus the sample blank (346 nm emission); E _STD is the fluorescence measurement minus the standard blank (346 nm emission); W _SMP is the sample weight (mg); W _STD Is the standard weight (mg); V _SMP is the sump dilution (mL); V _STD is the standard dilution (mL); FC is the correction factor (see Table 1).

Example 4
Validation test of total protein assay by fluorescence spectroscopy The performance characteristics of the fluorometric determination of the total protein amount of API released from the pancrelipase composition (Zenpep® formulation) in the elution test are as follows: specificity, linearity The results are summarized in Table 2, evaluated by proof of integrity, accuracy, precision, limit of quantification, sample and standard stability, extraction integrity in standard preparation.

  The validation data obtained here show that the fluorometric method proposed for determining the total protein amount in the dissolution test of the pancrelipase dosage form (Zenpep® formulation) is suitable for the intended use .

Example 5
Pancrelipase beads (Zenpep® minitab elution profile: total protein content by fluorescence spectroscopy (non-specific assay), proteolytic activity by protease assay (enzyme-specific assay), and Lipolytic activity by lipase assay (enzyme specific assay) This dissolution test is performed by sampling 2.5 mL aliquots at 10, 12, 15, 18, and 30 minutes according to the method described above (Examples). (See 2) No solvent exchange during the test To compare the elution profiles with the f2 test, a comparison of the three methods was compared with the SUPAC approach (GUI forance industry “SUPAC MR: Modified release solid oral dosa”). e forms.Scale-up and postapproval changes: chemistry, manufacturing, and controls, in vitro dissolution testing, and in vivo bioequivalence documentation "Center for Drug Evaluation and Research (CDER), for each .3 one of the measurement method carried out according to September 1997) To generate the required number of data, 12 independent samples (sample = amount of pancrelipase beads, Zenpep® minitab corresponding to 11,200 UI), 4 times in groups of 3 samples at a time To analyze.

Example 5.1 Dissolution Profile of Pancrelipase Beads (Zenpep® Mini Tablets) by Protease Assay The respective dissolution values and overall average at each time point tested are summarized in Table 3. The average curve is shown in FIG.

Example 5.2. Dissolution profile of pancrelipase beads (Zenpep® mini-tablets) by lipase assay The respective dissolution values and overall average at each time point tested are summarized in Table 4. The average curve is shown in FIG. In the calculation, a correction factor of 1.125 is used to correct lipase degradation during the dissolution test.

Example 5.3 Dissolution Profile of Pancrelipase Beads (Zenpep® Mini Tablets) by Fluorometric Determination of Total Protein Amount The respective dissolution values and overall average at each time point tested are summarized in Table 5. The average curve is shown in FIG.

Example 5.4 Comparison of Dissolution Profiles Obtained by Three Assays Table 6 shows the average dissolution data of pancrelipase beads (Zenpep® mini-tablets) measured at each time point by three assay methods. To summarize.

  The three elution profiles overlap almost completely as shown in the comparison diagram of FIGS. 4a-c, in particular, the average curves of the lipase assay and the fluorescence assay can be completely superimposed and the protease average curves do not match Is only for endpoints exhibiting a value> 100%. The CV of each series of data is also very similar in the three measurement methods, and the value of the fluorescence assay is small.

  What is currently used (lipase assay) using the SUPAC approach (similarity test f2 to compare product elution profiles) used to assess the equivalence of drug product performance before and after the change And the equivalent of the other enzyme-specific measurement (protease assay) and the newly proposed method for fluorescence quantification of total protein in the dissolution test of Zenpep® formulations.

In order to apply the similarity test f2 on the dissolution profile, FDA guidelines (Guidance for Industry, “SUPAC MR: Modified release solid and oral dosage forms. Scale-up and postproventive changes: vivo bioequilience document "Center for Drug Evaluation and Research (CDER), September 1997; Guidance for Intrusion-dissolution testin. of Immediate Release Solid Oral Dosage Forms, Center for Drug Evaluation and Research (CDER), August 1997) shows some of the conditions to be satisfied by the following:
a. Use the average elution value (n = 12) of both curves at each time interval,
b. One-time measurements should be considered after the 85% elution point,
c. The average difference between elution profiles at any sampling time should not exceed 15%,
d. To allow the use of average data, the coefficient of variation (%) at the initial time should not exceed 20% and at other times should not exceed 10%.

  In this elution data, requirements a, b and c are satisfied. However, a CV value greater than the CV value that allowed (d) was observed at the first three time points (10 minutes, 12 minutes, 15 minutes) of all measurement methods evaluated. The high variability observed at these time points is that the first sampling time (10 minutes) takes into account that 100% release of the formulation is obtained in a very short time (30 minutes) under the dissolution test conditions. This can be explained by the very low concentration of the analyte and the intrinsic variability of the formulation over a short range of elapsed time after the 12 and 15 minute time points.

  Based on the above judgment, the f2 test is applied anyway, assuming that similar variability observed by the three measurement methods showed sufficient overlap without significantly changing the resulting average curve. Then, in order to verify whether the f2 test can be passed even if data that completely satisfies the SUPAC requirements is used, further evaluation is performed in consideration of the last three time points (15 minutes, 18 minutes, and 30 minutes).

Example 5.4.1. Comparison: fluorimetric content and lipase assay The f2 test on the elution profile of the fluorimetric and lipase assay (standard) showed 87.4% similarity between the two curves when considering all time points, compared to the last As a result, the similarity was 83.3%.

Example 5.4.2. Comparison: Fluorometric and protease assay of total protein content The f2 test on elution profiles of the fluorometric and protease assay (standard) showed 72.3% similarity between the two curves when all time points were considered On the other hand, the similarity was 68.6% when calculated for the last three time points. In general, an f2 value greater than 50 (50-100) guarantees the identity or equivalence of the two curves, and thus the identity or equivalence of the test performance. Thus, according to the results obtained by comparing the dissolution profiles by the f2 test, the fluorescence quantification of the total protein amount is not limited to the measurement of API release in the dissolution test of the pancrelipase preparation (Zenpep® preparation). It can be said that it is equivalent to an enzyme-specific method.

Example 6
Validation Data Comparison To complete the comparison between the new fluorescence test and the known recognized lipase activity enzyme assay (USP method), Table 7 is shown.

Example 7
Dissolution test for pancrelipase beads by fluorescence spectroscopy.
In addition, a fluorescence analysis method as described in the above example is performed using another pancrelipase formulation marketed under the name Creon® with different formulation titers. The results are compared to those obtained with pancrelipase beads marketed as Zenpep® and are reported in FIG. The difference in behavior is consistent with the difference in formulation titer and the difference in particle surface area of the two formulations (Zenpep®, Creon®). Two different bead sizes are used for Zenpep® with different titers, with a smaller size (average diameter about 1.8 mm) showing a faster elution profile among 5,000 UI capsules. On the other hand, the larger size (average diameter about 2.4) is used in 20,000 UI capsules. Creon® beads have an average diameter of about 1 mm, but are significantly more irregular than Zenpep® beads, and thus show a faster elution profile than Zenpep®, but with less reproducibility between batches (All Creon® titers use the same granules).

Example 8
Dissolution test combined with gastro-resistance test: FL test and GR test FL test: Dissolution test by fluorescence measurement Dissolution test of 800 mL in each vessel of basket equipped with USP Apparatus 1-basket Solvent is added and the elution solvent (acidic stage) is equilibrated at 37 ° C. Weigh out an amount of pancrelipase beads equivalent to 11,200 USP lipase units (10 capsules of Zenpep® minitabs or microtabs) and transfer to each basket of Apparatus 1. The apparatus is operated at 100 rpm. After 1 hour, the baskets are removed from the solvent, rinsed with a few milliliters of water, and the contents of each basket contain 800 mL of enteric-stage solvent in an elution tank equipped with a paddle device (USP Apparatus 2) at 37 ° C. Transfer to elution vessel. The apparatus is operated at 100 rpm. After 30 minutes, 10 mL of the solution under test is removed, transferred to a test tube, and allowed to equilibrate at room temperature. Further dilution with enteric stage solvent to obtain the appropriate concentration (approximately 0.3 USP units / mL). Store the solution in an ice-water bath and shake by hand before reading. The diluted solution is read with a fluorescence spectrometer using the following operating parameters: quartz cuvette with 1 cm optical path length; excitation wavelength: 280 nm; emission wavelength (measurement) = 346 nm; excitation slit: = 6.0.

  The target concentration of marker (API = pancrelipase; 100% release) in the diluted test solution is 0.3 USP lipase units / mL.

  Standard solutions are prepared as described in Example 1 and stored in an ice-water bath with stirring until reading.

  The amount of drug released from the pancrelipase solid composition is calculated as follows.

The bulk minitab / microtab is calculated by the following formula.

式中、ＬＣはサンプルの蛍光測定値（３４６ｎｍの発光）であり；ＬＳは標準の蛍光測定値（３４６ｎｍの発光）であり；ＰＳは標準の重量（ｍｇ）であり；ＰＣはサンプル重量（ｍｇ）であり；ＵＳは標準の力価（リパーゼＵＳＰ単位／ｍｇ、バッチリパーゼアッセイ）であり；ＰＭはカプセル内容物の平均重量（ｍｇ／カプセル）であり；ＵＬは個々の投薬単位中のリパーゼの表示内容物（ＵＳＰ単位／カプセル）であり；ＶＣはサンプルの希釈量（ｍＬ）であり；ＶＳは標準の希釈量（ｍＬ）である。 The capsule is calculated by the following formula.

Where LC is the fluorescence measurement of the sample (emission at 346 nm); LS is the standard fluorescence measurement (emission at 346 nm); PS is the standard weight (mg); PC is the sample weight (mg US is the standard titer (lipase USP units / mg, batch lipase assay); PM is the average weight of the capsule contents (mg / capsule); UL is the lipase in the individual dosage unit Display contents (USP units / capsule); VC is sample dilution (mL); VS is standard dilution (mL).

GR test: Gastroresistance test (by lipase assay)
800 mL of acidic solvent is placed in each vessel (USP Apparatus 1-basket) of the elution tank equipped with a basket apparatus, and then the elution solvent is equilibrated at 37 ° C. Weigh out an amount of pancrelipase beads equivalent to 11,200 USP lipase units (10 capsules of Zenpep® minitabs or microtabs) and transfer to each basket of Apparatus 1. The apparatus is operated at 100 rpm. After 1 hour, remove the basket from the solvent, immerse the basket in a 1,000 mL beaker containing 900 mL of cold purified water at 4 ° C., rinse the sample for a short time (about 5 seconds), and without changing the rinse solvent, This process is repeated three times. Quantitatively transfer the contents of each basket to a ceramic mortar and add 5-6 mL of cold purified water. Grind the sample until a complete dispersion of the sample is obtained, collect quantitatively in a 200 mL volumetric flask and rinse the mortar well. This process is repeated 2-3 times. Add purified water to final volume. Further dilution with cold purified water is performed to obtain the appropriate concentration (approximately 14 USP units / mL, the theoretical maximum concentration available assuming 100% gastronomy of the product). Sample solutions are prepared immediately prior to titration and stored in an ice-water bath with stirring.

  The standard solution is prepared as follows: USP pancreatine lipase RS in an amount corresponding to 6,000 USP units, or accurately weighted to the standard for measurement of pancrelipase, added to a ceramic mortar, about 5-6 mL Add purified water and then grind. Pour the mortar liquid into a 500 mL volumetric flask and rinse the mortar further. Repeat this operation 2-3 times. Add purified water to final volume. A standard solution is prepared immediately before titration and stored in an ice-water bath with stirring.

  The final concentration of the standard solution is about 12.0 USP unit lipase / mL.

  For the lipase assay: Pour 30 mL of substrate emulsion (37 ° ± 0.1 ° C.) into the jacketed vessel of the titrator and stir with a magnetic stirrer to maintain the temperature at 37 ° ± 0.1 ° C. 0.1N NaOH is added and the pH is adjusted to 9.20-9.23 by potentiometric titration. Add 1.0 mL of sample or standard, followed by 0.1 N NaOH for 5 minutes to maintain pH at 9.0.

式中、ＶＭＣはサンプルにより１分あたりに消化される０．１ＮのＮａＯＨの量（ｍＬ／分）であり；ＡＶＭＳは標準により１分あたりに消化される０．１ＮのＮａＯＨの平均量（ｍＬ／分）であり；ＰＣはサンプルの重量（ｍｇ）であり、ＰＳは標準の重量（ｍｇ）であり；ＤＣはサンプルの希釈量（ｍＬ）であり；ＤＳは標準の希釈（ｍＬ）であり；ＵＳは標準の力価（リパーゼＵＳＰ単位／ｍｇ）であり；ＵＣはバッチリパーゼアッセイ（ＵＳＰリパーゼ単位／ｍｇ）である。 The calculation is performed using the following formula:

Where VMC is the amount of 0.1 N NaOH digested per minute by the sample (mL / min); AVMS is the average amount of 0.1 N NaOH digested per minute by the standard (mL PC is the weight of the sample (mg), PS is the standard weight (mg); DC is the sample dilution (mL); DS is the standard dilution (mL) US is the standard titer (lipase USP units / mg); UC is the batch lipase assay (USP lipase units / mg).

Acceptance criteria according to USP, <711> dissolution-Delayed Release forms (acid stage), Acceptance Table 3 are as follows:
Level 1 acceptance criteria: GR% of individual units is 90% or less;
Level 2 acceptance criteria: The average value of GR for 12 units (6 for Level 1 and 6 for Level 2) is less than 90%, each unit being less than 75%
Level 3 acceptance criteria: The average value of GR for 24 units (6 units for level 1, 6 units for level 2 and 12 units for level 3) is less than 90%, and each unit is less than 75%.

Based on all the data and considerations reported above, the fluorescence measurement of total protein as a marker of digestive enzymes released in the elution test of pancrelipase beads is based on currently accepted compendia based on lipase activity assays. This is a reliable and advantageous measurement method that can replace the above measurement method.

Claims (22)

In the method of measuring the amount of digestive enzyme released from the pancrelipase solid composition into the elution solvent, fluorescence spectroscopy is used to measure the amount of digestive enzyme released from the composition into the elution solvent. A method comprising the step of using. The method of claim 1 , wherein the pancrelipase solid composition is an enteric pancrelipase composition. 3. The method according to claim 1 or 2, wherein the elution solvent is water, HCl solution, artificial gastric fluid, buffer solution, artificial intestinal fluid, aqueous solution or buffer solution containing at least one surfactant. 4. The method according to claim 1, 2 or 3 , wherein (a) releasing the digestive enzyme from the solid pancrelipase composition into the elution solvent; and b) reading the fluorescence and digesting enzyme in the solvent. Measuring the amount of the method. The method according to claim 1, 2, 3 or 4, wherein the elution solvent method characterized by comprising at least two solvents are used over time. 6. The method of claim 5 , wherein a) adding the solid pancrelipase composition to a first elution solvent, b) transferring the suspension to a second elution solvent, and c) the digestion. Causing the release of the enzyme; d) sampling an aliquot of the eluting solvent; e) reading the fluorescence at 346 nm; and f ) calculating the amount of digested enzyme released. Feature method. 7. The method of claim 6 , wherein the first eluting solvent is an aqueous solvent having a pH of about 1 to about 4.5 and the second eluting solvent is an aqueous buffer having a pH greater than about 5. A method characterized by that. 8. The method of claim 6 or 7 , wherein the first eluting solvent is an aqueous solvent having a pH of about 1 to about 2. 9. The method of claim 6, 7 or 8 , wherein the second eluting solvent is an aqueous buffer having a pH of about 5.5 to about 6.8. 10. The method of claim 6, 7, 8 or 9 , wherein the first eluting solvent is an aqueous solvent having a pH of about 1.2 and the second eluting solvent is an aqueous buffer having a pH of about 6. A method characterized by being. The method according to claim 6, 7, 8, 9 or 1 0, the pancrelipase compositions about 750USP lipase units in total, about 3,000USP lipase units, about 4,200 USP lipase units, about 5,000 USP lipase units, about 6,000 USP lipase units, about 10,000 USP lipase units, about 10,500 USP lipase units, about 15,000 USP lipase units, about 16,800 USP lipase units, about 20 USP lipase units, about 21,000 USP lipase units, about 24,000 USP lipase units, or about 25,000, or about 40,000 USP lipase units or multiples thereof, or about 5,000 PhEur lipase units, or about 10,000 PhEur. Lipase A unit, or about 15,000 PhEur lipase unit or about 20,000 PhEur lipase unit or about 30,000 PhEur lipase unit, or about 40,000 PhEur lipase unit, or a multiple thereof. The method according to claim 7, 8, 9, 10 or 1 1 in combination with an enteric (gastroresistance) test, enteric of the composition of the aqueous solvent A method characterized in that residual lipase activity is measured by determining with a specific lipase assay. 13. The method of claim 12 , wherein the aqueous solvent has a pH of about 1 to about 2. The method of claim 12 , wherein the aqueous solvent has a pH of about 1.2. 15. The method according to claim 13 or 14 , wherein the measurement by fluorescence spectroscopy of the amount of digestive enzyme released from the solid composition into an elution solvent is performed before or after the gastroenteric test. A method characterized by. 16. The method according to claim 13, 14 or 15 , wherein the measurement by fluorescence spectroscopy of the amount of digestive enzyme released from the solid composition into the eluting solvent is from at least two solvents used over time. The method is performed in an elution solvent. 17. The method of claim 16 , wherein the first eluting solvent is an aqueous solvent having an acidic pH of about 1 to about 4.5. 18. The method of claim 16 or 17 , wherein the first eluting solvent is an aqueous solvent having a pH of about 1 to about 2. 19. The method of claim 16, 17 or 18 , wherein the second eluting solvent is an aqueous buffer having a pH greater than about 5. 20. The method of claim 16, 17, 18 or 19 , wherein the second eluting solvent is an aqueous solution having a pH of about 5.5 to about 6.8. 21. The method of claim 16, 17, 18, 19 or 20 , wherein the first eluting solvent is an aqueous solvent having a pH of about 1.2 and the second eluting solvent is an aqueous buffer having a pH of about 6. A method which is a liquid. 24. The method of claim 12, 13, 14, 15, 16 , 17 , 18, 19 , 20 or 21 , wherein the pancrelipase composition comprises a total of about 750 USP lipase units, about 3,000 USP lipase units, about 4 , 200 USP lipase unit, about 5,000 USP lipase unit, about 6,000 USP lipase unit, about 10,000 USP lipase unit, about 10,500 USP lipase unit, about 15,000 USP lipase unit, about 16,800 USP lipase unit, about 20, 000 USP lipase units, about 21,000 USP lipase units, about 24,000 USP lipase units, about 25,000 USP lipase units, or about 40,000 USP lipase units or multiples thereof, or about 5,000 PhEur lipase units, or about 1 A method characterized by having a 000PhEur lipase units or about 15,000PhEur lipase units or about 20,000PhEur lipase units or about 30,000PhEur lipase units or about 40,000PhEur lipase units or a multiple thereof,.

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